RESEARCH ARTICLE Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387Please cite this article in press as: Susena S. et al., A Novel validated RP-HPLC method for theestimation of Liraglutide in bulk and Parenteral Dosage form. American Journal of PharmTech Research2013.A Novel validated RP-HPLC method for the estimation ofLiraglutide in bulk and Parenteral Dosage formS. Susena*1, K. Vanitha Prakash1, T.Radika1, R.Tejaswini1,E.Manasa11. SSJ College of Pharmacy, V.N. Pally, Gandipet, Hyderabad-500075(India).Department: Pharmaceutical Analysis & Quality assurances)ABSTRACTA simple, accurate, precise and rapid reversed-phase high performance liquid chromatographic(RP-HPLC) method has been developed and subsequently validated for the estimation ofLiraglutide in bulk and Parenteral Dosage form. The proposed method is based on the separationof drugs in reversed-phase mode using Waters HPLC 22695 model, Inertsil ODS column (250 x4.6 mm, 5µm particle size).The optimum mobile phase consisted of methanol: phosphate bufferin the ratio of 85:15 v/v(Phosphate buffer pH 5.5) was selected as a mobile phase, flow rate of1.0 ml/min and UV detection was set at 246 nm. The retention time was 3.25.The method wasvalidated according to ICH guidelines. It was found to be accurate and reproducible. Linearitywas obtained in the concentration range of 20-80 μg/ml . The percentage RSD for precision andaccuracy of the method was found to be less than 2%. The proposed method can be successfullyapplied in the quality control of bulk and pharmaceutical dosage forms.Keywords:, Liraglutide ,RP-HPLC, Validation*Corresponding Author Email: email@example.comReceived 10 April 2013, Accepted 14 May 2013Journal home page: http://www.ajptr.com/
Susena et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387www.ajptr.com 2INTRODUCTIONLiraglutide is a long-acting glucagon-like peptide-1 (GLP-1) analog that has been developed byNovo Nordisk for the treatment of type2 diabetes. The product was approved by the EuropeanMedicines Agency (EMA) on July 3, 2009, and by the U.S. Food and Drug Administration(FDA) on January 25, 2010.Liraglutide intended to help lower blood sugar levels along with diet, exercise, and selected otherdiabetes medicines. It is not recommended as initial therapy in patients who have not achievedadequate diabetes control on diet and exercise alone. Liraglutide is an incretin mimetic. Thismeans that it mimics the actions of incretin hormones in the body. As an incretin mimetic,liraglutide increases insulin production in response to meals and decreases the amount of glucose(sugar) that the liver produces. The medicine also slows the emptying of food from the stomachand decreases the amount of food that people eat.Figure 1: Structure of LiraglutideMATERIALS AND METHODApparatus:Waters HPLC 22695 series consisting 4 pump, Auto sampler, equipped with PDA detector,thermostat column connected with waters (alliance) Empower software ,Inertsil ODS C18column (250x4.6mm, 5μm in particle size), Ohaus weighing balance and bath sonicator, borosilglass apparatus were used for experimental purpose.Chemicals and reagents:Liraglutide pure drug was obtained as a gift sample from HETERO LABS LIMITED.Hyderabad. Methanol and phosphate Buffer (PH-5.5) were purchased from S.D. Fine (P) Ltd.Mumbai. All chemicals and reagents used were of analytical HPLC grade.Preparation of mobile phase:Isocratic elution with Methanol: phosphate Buffer (PH-5.5) 85:15(V/V) was used at a flow rateof 1.0ml/min. The mobile phase was prepared freshly and degassed by sonicating for 5 minbefore use.
Nagariya et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-33873 www.ajptr.comPreparation of standard and test solutionsLiraglutide standard stock solution:10 mg Liraglutide was accurately weighed and dissolved in 10 ml methanol then transferred to a10 ml volumetric flask sonicate it for 5 min, finally volume was made up to the mark withmethanol to make 1000μg/ml stock solution.Working solution:1ml of the above stock solution was pipetted into a 10ml volumetric flask and diluted up to themark with HPLC grade Methanol. The contents were mixed well and filtered through 0.45μmnylon filter paper to get this stock solution (100 μg/ml).Preparation of sample solution:For the analysis of the dosage form, Liraglutide in injection form manufactured by novonordiskLtd. was Selected. By using Liraglutide injection 1.7ml(i.e.10mg)of solution is transferred to10ml volumetric flask finally volume was made up to the mark with methanol to make1000μg/ml sample solutionMethod validationThe method was validated in accordance with ICH guidelines17. The parameters assessed werelinearity, accuracy, and limit of detection (LOD), limit of quantification (LOQ), precision,reproducibility and robustness.RESULTS AND DISCUSSIONSLinearityThe calibration curve obtained by plotting peak area against concentration showed linearity inthe concentration range of 20-80 μg/ml Linear regression data for the calibration curves aregiven in Figure 2.Figure 2: Linear regression data for the calibration curve0100000020000003000000400000050000006000000700000080000000 20 40 60 80 100
Susena et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387www.ajptr.com 4AccuracyThe % mean recovery obtained for Liraglutide was 101% .The %RSD is less than 2, results weregiven in Table 1.Table 1: Accuracy data for proposed methodSpiked level ofdrug (%)Amount of drugadded (μg/ml)Amount of drugfound (μg/ml)%Recovery50 20 20.29 105.0100 40 40.2 101.0150 60 60.7 98.0Detection limit and quantification limitLOD for Liraglutide was 0.24 μg/ml respectively, while LOQ was 0.72 μg/mlPrecisionResults for repeatability expressed as %RSD, results were given in Table 2. The low values of%RSD indicate that the method is precise. Reproducibility was checked by analyzing thesamples by another analyst using same instrument and same laboratory. There was no significantdifference between the %RSD values, which indicates that the proposed method wasreproducible, results were showed in Table 2.Table 2: Precision of the proposed HPLC methodConc. of Liraglutide(40 μg/ml)Peak area of LiraglutideIntra-day Inter-dayInjection-1 3218338 3318621njection-2 3230606 3326451Injection-3 3250591 3412131Injection-4 3242511 3332153Injection-5 3257321 3365143Average 3239873.4 3350899.8Standard Deviation 7802.8 14435.5% RSD 0.2 0.4Table 3: Results of robustness for proposed methodFactor Level Retention time AsymmetryA: Flow rate (ml/min)0.8 -0.2 3.90 1.341.0 0 3.25 1.321.2 +0.2 2.89 1.28%RSD 0.3 0.7B: % of methanol (ml)84 -1 3.70 1.3485 0 3.26 1.3286 +1 3.10 1.28%RSD 0.2 0.7
Nagariya et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-33875 www.ajptr.comDetection limit and quantification limitLOD for Liraglutide was 0.24 μg/ml respectively, while LOQ was 0.72 μg/mlRobustnessThere was no significant change in the peak areas and retention times of Liraglutide, when thecomposition of mobile phase ±1ml and flow rate was varied by ±0.2 ml. The results are showedin Table 3.SpecificityNo interference from any of the excipients was found at retention times of the examined drugs.In addition, the chromatogram of each drug in the sample solution was found identical to thehromatogram received by the standard solution at the wavelengths applied. These resultsdemonstrate the absence of interference from other materials in the pharmaceutical formulationsand therefore confirm the specificity of the proposed method.System suitabilityThe acceptance criteria are % RSD of peak areas and retention time less than 2%, theoreticalplates numbers (N) at least 4500 per each peak and tailing factors less than 1.5 for Liraglutide theresults are shown in the Table 4.Table 4: System suitability parametersParameters LiraglutideLinearity (μg/ml) 20.-80Correlation coefficient 0.998Theoretical plates 9248Tailing factor 1.32LOD (μg/ml) 0.24LOQ (μg/ml) 0.72Quantification of Liraglutide in parentral dosage formThe proposed method was applied to the estimation of Liraglutide in parentral dosage form. Theresults of the assay 99.84±0.24%, of label claim of the injection. The assay results showed thatthe method was selective for the estimation of Liraglutide without interference from theexcipients used in the injection form. The results are shown in the Table 5.Table 5: Results of sample analysis for proposed methodBrand name Analyte Label claim perinjection (mg)% Analyteestimated(mean±SD)%RSDVICTOZA Liraglutide 10 99.84±0.24 0.193In order to achieve the estimation of the Liraglutide initial trials was performed with theobjective of selecting adequate and optimum chromatographic conditions. Parameters, such as
Susena et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387www.ajptr.com 6ideal mobile phase and their proportions, detection wave length and concentrations of thestandard solutions were carefully studied. Several solvents were tested in varying proportions.Finally, a mixture of methanol: Phosphate buffer (85:15 v/v) was selected as the optimum mobilephase. The optimized chromatographic conditions were selected based on sensitivity, retentiontimes and peak shape. The method was validated in terms of linearity, accuracy, precision, LOD,LOQ, robustness and specificity as per ICH guidelines. The accuracy data shows that the methodis accurate within desired range. The LOD and LOQ values were low which indicates that themethod is sensitive. The method was robust as minor changes in the chromatographic parametersdid not bring about any significant changes in peak area and retention times of LiraglutideName RetentionTimeArea % Area Height USPTailingSymmetryFactorUSP PlateCountLiraglutide 3.259 3250591 100.00 374481 1.329972 1.339972 9283Figure. 3: Typical chromatogram of Liraglutide standardCONCLUSIONA validated RP-HPLC method has been developed for the determination of Liraglutide ininjection form. The proposed method is simple, rapid, accurate, precise and specific. Itschromatographic run time of 10 min allows the analysis of a large number of samples in shortperiod of time. Therefore, it is suitable for the routine analysis of Liraglutide in pharmaceuticaldosage form. The limit of detection for Liraglutide was found to be 0.24 μg/ml and the limit ofquantification was found to be 0.72 μg/ml. It proves the sensitivity of method.REFERENCES1. http://www.novonordisk.com/science/about_rd/quarterly_rd_update.asp Oct 2008 Incesults of LEAD 6 extension2. http://money.cnn.com/news/newsfeeds/articles/marketwire/0580389.htm January 2009Liraglutide-3.259AU0.000.020.040.060.080.100.120.22.214.171.124Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Nagariya et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-33877 www.ajptr.com3. Hirschler, Ben (January 13, 2010). "UPDATE 1-Novo starts tests on pill version ofVictoza drug".Reuters.4. "Sector Snap: New Diabetes Drugs under FDA Review". Forbes. Retrieved March 27,2009.5. http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/Endocr inologicandMetabolic Drugs Advisory Committee/UCM148659.pdf6. http://www.drugs.com/fda/victoza-liraglutide-rdna-origin-rems-risk-thyroid-c-cell-tumors-acutepancreatitis- 12979.html7. http://diabetes.webmd.com/news/20080924/new-diabetes-drug-liraglutide-works Sept20088. http://care.diabetesjournals.org/cgi/content/abstract/32/1/84 "Efficacy and SafetyComparison of Liraglutide, Glimepiride, and Placebo, All in Combination withMetformin, in Type 2 Diabetes" Diabetes Care. Oct 20089. "Novo Nordisk Limited, Eli Lilly and Company Limited, Grünenthal Ltd and NappPharmaceuticals Limited named in advertisements". Prescription Medicines Code ofPractice Authority (PMCPA). Retrieved 2011-02-07.10. http://www.mmm-online.com/novo-reps-to-spotlight-weight-loss-for-victozalaunch/Article/163167/ | Medical Media & Marketing; Novo reps to spotlight weight loss forVictoza launch; 2/4/2010; Ben Comer.